Selective binding to closely related protein isoforms represents a particularly challenging problem in the search for molecules that might control cell signaling. This is nowhere more true than in the disruption of constitutively activated Signal Transducer Activators of Transcription (STAT) proteins as a viable anti-cancer therapy. Constitutive activation in numerous cancers, including leukemia and lymphomas, has provided evidence of a relationship between aberrant STAT activation and oncogenesis. As a result, the emerging significance of STAT signaling in the development of human cancers makes it an excellent target for new therapeutic intervention.
The STATs are a family of cytoplasmic proteins important in cell proliferation, differentiation, apoptosis and survival. STAT activated dimers have been shown to interact with specific promoter regulatory elements to induce target gene transcription. STATs are triggered through extracellular cytokine and growth factor stimulation resulting in receptor dimerization and activation. Phosphorylation of a crucial tyrosine residue provides binding sites for the recruitment of monomeric, non-phosphorylated STAT proteins via their Src homology 2 (SH2) domain. Receptor-bound STAT3 is then tyrosine phosphorylated by receptor and/or nonreceptor tyrosine kinases such as Src and JAK. Phosphorylated STAT proteins are then released from the receptor, and dimerization occurs through reciprocal phosphotyrosine-SH2 interaction. STAT dimers immediately translocate to the nucleus and bind with promoter regulatory elements. In normal functioning cells STAT activation is transitory and tightly regulated. However, aberrant STAT activation leads to the up-regulation of oncogenic pathways through dysregulated growth, suppression, angiogenesis and survival. The alleviation of irregular STAT signaling through dimerization inhibition provides a focused target for molecular intervention. Suppression of homo and hetero-dimerization through specific peptide sequences interacting with STAT has served to establish the structural attributes required for SH2 domain-inhibitor complementarity and further development of anti-cancer agents. Critical for their roles in cell biology the seven individual isoforms of STAT exhibit different functional properties. For example, STAT1 deficient mice display an impaired response to interferons, are susceptible to viral or bacterial pathogens and spontaneously develop tumors. Also, constitutively activated STAT1 signaling is present in a number of human cancers although its role in oncogenic pathways has yet to be fully elucidated. In contrast to STAT1, inhibition of persistant STAT3 activation by blocking tyrosine kinase activity has been repeatedly associated with tumor selective growth suppression and cell death.
It has been previously reported that the synthesis of STAT3 homodimerization inhibitors through the use of the peptide PY*LKTK (Y*=phosphotyrosine), which corresponds to the core of the native C-terminal STAT3 SH2 domain binding sequence GSAAPY*LKTKFIC. (see Turkson, J., Ryan, D., Kim, J. S., Zhang, Yi, Chen, Z., Haura, E., Laudano, A., Sebti, S., Hamilton, A. D., Jove, R., J. Bio. Chem., 2001, 276, 45443; Turkson, J., Kim, J. S., Zhang, S., Yuan, J., Huang, M., Glenn, M., Haura, E., Sebti, S., Hamilton, A. D., Jove, R., Mol. Canc. Therap., 2004, 3, 261; and Becker, S., Groner, B., Muller, C. W., Nature, 1998, 394, 145; which are incorporated herein by reference). Further optimization through the synthesis of a focused tripeptide library identified the importance of the central Y*L residues for inhibitory activity. Specific STAT selectivity was associated with functional group substitution of the peptide termini. The native binding sequence from STAT1 (PKGTGY*IKTELIS) shows marked variation from that of STAT3 near the termini of the region investigated, corresponding well with the selectivities observed among prepared compounds. By testing with a range of substituents of varying polarity, size and orientation it was shown that a phenyl nitrile substituent at the western Y* N-terminus as being crucial in the series for enhanced inhibition of STAT3 dimerization.